High level expression of recombinant proteins in host cells such as E. coli often leads to accumulation of proteins as insoluble aggregates in vivo as inclusion bodies. Inclusion bodies are dense aggregates of misfolded polypeptides devoid of bioactivity that need elaborate solubilization/refolding protocols to achieve a native conformation. The formation of inclusion bodies is mainly attributed to the over-expression of proteins in a cell lacking required accessories for its folding to native form. Endogenous proteins when over-expressed also accumulate as inclusion bodies. There is no direct correlation between the propensity of the inclusion body formation of a certain protein and its intrinsic properties, such as molecular weight, hydrophobicity, folding pathways and so on. In case of proteins having disulfide bonds, formation of protein aggregates as inclusion bodies is anticipated since the reducing environment of bacterial cytosol inhibits the liberation of disulfide bonds. It is desirable to recover these proteins from the cells for maximum recovery of bioactive proteins.
Proteins expressed as inclusion bodies are currently solubilized by the use of high concentration of chaotropic agents such as urea, guanidine hydrochloride, and thiocyanate salts, detergents such as SDS, N-cetyl trimethyl ammonium chloride, and sarkosyl (sodium N-lauroyl sarcosine). The soluble denatured proteins are then refolded to their native state after removing the chaotropic agents or other salts by dialyzing the proteins in buffers containing reducing and oxidizing agents. Renaturation of recombinant proteins from inclusion bodies into bioactive form is cumbersome, results in low recovery of the final product and also accounts for the major cost in overall production of recombinant proteins. However in the case where simple high yielding protein refolding process is developed for the aggregated recombinant proteins, high level expression of proteins as inclusion body provides a straightforward strategy for the cost-effective production of therapeutic proteins. Significant features of protein aggregates in inclusion bodies are the existence of native-like secondary structure of the expressed protein and their resistance to proteolytic degradation. The aggregation leading to inclusion body formation has also been reported to be due to specific intermolecular interaction among a single type of protein molecules. The formation of inclusion bodies thus facilitates the easy isolation and recovery of the expressed proteins in denatured form. Loss during recovery of protein from inclusion bodies is compensated by high initial level of expression.
It has also been reported that protein aggregation in inclusion bodies is a reversible process and inclusion bodies are resistant to proteolytic degradation and peptide degradation process by enzymes occurs as a cascade in situ. Solubilization profile of inclusion bodies in different buffers gives an idea about the dominant forces involved in protein aggregates during high level expression of recombinant protein as inclusion bodies. Such information can thus be exploited to develop mild solubilizing buffer, which will protect the native-like secondary structure of the protein during solubilization. Presence of other contaminating proteins has negative effect on overall yield of the bioactive protein during refolding of denatured protein. As aggregation is the major factor responsible for the reduced yield of bioactive protein from the inclusion bodies, it is desirable to develop soluilization process which does not unfold the protein completely.
Some workers have tried to increase the overall yield of purified bioactive proteins from the inclusion bodies by trying to protect the existing secondary structure of the proteins during solubilization and the refolding is carried out in such a way that interaction between partially folded polypeptide intermediates is minimized. This is achieved using detergents, high pH, use of high pressure for solubilization of protein aggregates. However all these above methods have their inherent disadvantages. High pH treatment sometimes tends to denature the proteins, it is difficult to remove the detergents after solubilization and high pressure does not completely solubilize the protein aggregates. Most of the time, the aggregation leading to inclusion body formation is predominantly due to hydrophobic interaction and due to mixed disulfide bond formation.
It would be ideal if the solubilizing agent has the ability of disrupting both hydrophobic interaction and disulfide bond formation resulting in solubilization of the inclusion body protein. Having worked on this line for long, the Applicant has developed a novel process that uses a novel denaturating solution for solubilization and recovery of inclusion body proteins in high yield from host cells.